HMG-CoA reductase is a potential therapeutic target for migraine: a mendelian randomization study

Statins are thought to have positive effects on migraine but existing data are inconclusive. We aimed to evaluate the causal effect of such drugs on migraines using Mendelian randomization. We used four types of genetic instruments as proxies for HMG-CoA reductase inhibition. We included the expression quantitative trait loci of the HMG-CoA reductase gene and genetic variation within or near the HMG-CoA reductase gene region. Variants were associated with low-density lipoprotein cholesterol, apolipoprotein B, and total cholesterol. Genome-wide association study summary data for the three lipids were obtained from the UK Biobank. Comparable data for migraine were obtained from the International Headache Genetic Consortium and the FinnGen Consortium. Inverse variance weighting method was used for the primary analysis. Additional analyses included pleiotropic robust methods, colocalization, and meta-analysis. Genetically determined high expression of HMG-CoA reductase was associated with an increased risk of migraines (OR = 1.55, 95% CI 1.30–1.84, P = 6.87 × 10−7). Similarly, three genetically determined HMG-CoA reductase-mediated lipids were associated with an increased risk of migraine. These conclusions were consistent across meta-analyses. We found no evidence of bias caused by pleiotropy or genetic confounding factors. These findings support the hypothesis that statins can be used to treat migraine.


GWAS
Genome-wide association study MR Mendelian randomization HMGCR 3-Hydroxy- Migraine, a common affliction endured by several people in headache clinics, is characterized by recurrent moderate-to-severe throbbing pain 1 .It affects ~ 12% of the global population and is the second most incapacitating ailment worldwide 2 .The deleterious impact of migraines on both well-being and quality of life cannot be underestimated because it imposes a substantial burden on a global scale 3 .Therefore, it is crucial to identify therapeutic targets for migraine to expand the available treatment options.

MR assumptions and study design
The selection of valid instrumental variables must satisfy three assumptions of MR analysis (Fig. 1) 17 .Given the observational association between circulating lipids and migraines, we first examined whether genetically predicted circulating lipids (LDL-C; apolipoprotein B, APOB; and total cholesterol, TC) were associated with migraines.Second, drug-targeted MR was performed to determine whether HMGCR expression in the blood affects migraines.Third, a colocalization analysis was performed to determine the presence of common genetic variants.Finally, to verify the observed associations, we assessed whether the levels of HMGCR-regulated LDL-C, APOB, and TC were associated with migraine.An overview of the study design is briefly outlined in Fig. 1.

Genetic variation for exposure and identifying instrumental variables
Summary-level GWAS data for circulating lipids were obtained from the UK Biobank.The circulating lipids included LDL-C (n = 431,167) 22 , APOB (n = 439,214) 23 , and TC (n = 342,508) 24 .Age, sex, and type of genotyping chip were adjusted as covariates in the GWAS analyses of individuals of European ancestry included in the UK Biobank.The criteria for the selection of instrumental variables were as follows: 1 genome-wide significance (P < 5 e−08); (2) linkage disequilibrium (LD) at R 2 < 0.001 within a 10,000-kb window based on the Europeanbased 1000 Genomes Project; (3) palindromic SNPs and SNPs with minor allele frequencies < 0.01 removed; (4) proxies not sought for instrumental variables not available in the outcomes; and (5) F-statistics of instrumental variables calculated using the square of the standard error 25 , with an F-value > 10 suggesting sufficient instrument strength 26 .
The available eQTLs for HMGCR (n = 31,684) can serve as genetic proxies for statins, and summary data for eQTLs were obtained from the eQTLGen Consortium 27 .The instrumental variables used in this study were consistent with those reported by Huang et al. 28 .The following inclusions were used: 1 genome-wide significant association (P < 5 e−08); (2) defined as cis-eQTLs located within ± 100 kb windows around the coding gene; (3) a minor allele frequency > 1%; and (4) demonstrating independent association (low LD clumping r 2 < 0.3).
Complementary analyses were conducted to verify the robustness of the associations obtained using cis-eQTLs as instrumental variables.Originally, we intended to use genetic instrumentation at the circulating HMGCR protein level (i.e., protein quantitative trait loci, pQTL) as a prerequisite for exposure validation.Unfortunately, we found no cis-acting pQTL that met these requirements.Therefore, considering that statins may affect the serum LDL-C, APOB, and TC levels, we selected these three circulating lipids as potential biomarkers 11,29,30 .We then selected genetic variants located within ± 100 kb of HMGCR (build GRCh37: chromosome 5:74,632,154-74,657,929) showing significant associations with LDL-C, APOB, and TC, respectively, at a genome-wide significance of P < 5 e−08, to serve as surrogates for statin therapy.LD was set to R 2 < 0.1 within a 100-kb window, using a European reference panel from the 1000 Genomes Project.This method of selecting instrumental variables was also used in previous studies 28,31 .Triglycerides and high-density lipoprotein cholesterol were excluded because no instrumental variables that met the above criteria were extracted.

Genetic variation for migraine
For the primary analysis, summary-level GWAS data for migraines were obtained from a meta-analysis conducted by the International Headache Genetics Consortium (IHGC), which included participants of European ancestry.The data were approved by a direct application and material transfer agreement 32 .The dataset used contained 48,975 migraine cases and 540,381 controls.Migraine cases were identified based on self-reported data or the International Classification of Headache Disorders.The cases included in this meta-analysis were adjusted for sex, age, and ancestry.The remaining details, including demographic characteristics, eligibility criteria, and ethical approval, can be found in the original article 32 .For replication analysis, summary statistics were obtained from the FinnGen study (nCase = 1,5905, nControl = 264,662, R8 release) 33 .

MR analysis
The primary analytical method for MR is random-effects inverse variance-weighted (IVW), which assumes that all SNPs are valid instruments, allows for balanced pleiotropy, and provides the most precise estimates 34 .Additional sensitivity analyses included the MR-Egger intercept test 35 , the weighted median test 36 , the radial MR test 37 , and the MR pleiotropy residual sum and outliers test (MR-PRESSO) 38 .The MR-Egger detected horizontal pleiotropy, with P > 0.05 indicating none.The MR-PRESSO and radial MR methods were employed to identify outliers.And visualization methods such as scatter plots and leave-one-out plots are also used to identify outliers.Heterogeneity among the different IVs was evaluated using Cochran's Q test.Burgess's online calculator was used to calculate the power of the MR estimates 39 .www.nature.com/scientificreports/

Bayesian colocalization analysis
To avoid the influence of LD or pleiotropy on MR findings, we performed a Bayesian colocalization analysis using the default parameters of the Coloc R package 40 .Bayesian colocation analysis was employed to assess the probability that the two traits (eQTL and migraine GWAS) shared the same causal variant 40,41 .We tested the posterior probabilities of five hypotheses: H0, not associated with any trait; H1/H2, associated with only one of the traits; H3, two traits having different causal variants; and H4, both traits having their causal SNPs and sharing the same SNP.We considered a posterior probability of hypothesis 4 (PPH4) > 0.8 (calculated by the Coloc.abfalgorithm) as strong evidence for colocalization.For visualization, we used the "locuscomparer" R package 42 .
The Bonferroni method was employed to adjust the significance threshold for four exposures, requiring P < 1.25 × 10 −2 .Estimates were considered significant in the MR analysis when at least the IVW method estimates were significant and the three different MR methods were considered consistent in direction.The association results are presented as odds ratios (OR) with 95% confidence intervals (95% CI).

Meta-analysis
We performed a random-effects meta-analysis of the results obtained from the IHGC and FinnGen datasets to produce a comprehensive analysis of causality.The R package "Metafor" was used for this.The significance levels for the heterogeneity tests and the effect values of the meta-analysis results were set to 0.05.

Additional positive control analysis
Given the common and beneficial use of lipid-lowering medications in coronary artery disease, the statin-medicated condition was used as a positive control to evaluate the reliability of the instruments.A total of 122,733 patients and 424,528 controls were included in the GWAS data for coronary artery disease 43 .

Ethical approval and consent to participate
This study used data from published studies.All original studies have been approved by the corresponding ethical review board, and the participants have provided informed consent.In addition, no individual-level data was used in this study.Therefore, separate ethical approval was not required for this study.

Results
Overall, 344 (LDL-C), 187 (APOB), and 259 (TC) SNPs were included in the analysis of the association between circulating lipids and migraine (Additional file 1: Supplementary Table 1).Two-sample MR analysis revealed no association between migraine and any of the three circulating lipids (Supplementary Tables 2-4).Seven eligible cis-eQTLs were included in the drug-targeted MRI analysis (Supplementary Table 5).From the GWAS summarylevel data, 18, 10, and 11 SNPs within or near the HMGCR region were associated with LDL-C, APOB, and TC, respectively (Supplementary Table 5).The F-statistic of all included instrumental variables was greater than 10, indicating the absence of weak instrumental variable bias.
As shown in Fig. 2, the primary analysis of migraine data from the IHGC revealed that genetically predicted expression of HMGCR was associated with increased risk of migraine (OR = 1.55, 95% CI 1.30-1.84,P = 6.87 × 10 −7 ).The replication study using data from FinnGen produced similar results (OR = 1.38, 95% CI 1.14-1.67,P = 7.38 × 10 −4 ).Both sensitivity analyses yielded similar estimates, and in the same direction (Supplementary Table 6).This finding indicated that HMGCR inhibitors may reduce the risk of migraine susceptibility.No statistically significant heterogeneity or horizontal pleiotropy was observed (Supplementary Table 6).When causal variants were present, Bayesian colocalization analysis using data from the IHGC suggested that HMGCR and migraine shared the same variants (Coloc.abf-PPH4= 0.97, Fig. 3) (Supplementary Table 7).
The genetically determined expression of HMGCR and the levels of LDC-C, APOB, and TC modified by HMGCR were associated with an increased risk of coronary artery disease (Fig. 5; Supplementary Table 8).As a reference for the positive control analyses, this result also increased the credibility of the included instrumental variables and confirmed the efficacy of the selected instruments.

Discussion
Consistent results obtained from a rigorous MR analysis indicated that HMGCR expression and the circulating levels of three lipids (LDL-C, APOB, and TC) adjusted by HMGCR were significantly associated with an increased risk of migraine.These findings strongly suggest that HMGCR inhibitors hold promise as potential protective medications against migraines.In line with the prior study by Bi et al. it was found that HMGCR could potentially serve as a therapeutic target for migraines 45 .The merits of our study include: firstly, the inclusion of a greater number of cis-eQTLs that could influence HMGCR gene expression; secondly, the GWAS studies for migraines encompassed larger sample sizes and a greater number of migraine cases; thirdly, both the discovery and replication analyses indicated that a genetically determined high expression of HMGCR was correlated with an elevated risk of migraines.Nevertheless, this association did not seem to be directly linked to the total circulating levels of LDL-C, APOB, or TC, as we did not identify any significant correlation between these lipid levels and the risk of migraine.In a recent meta-analysis examining the association between circulating lipids and migraine, individuals with migraine exhibited notably elevated levels of blood cholesterol, triglycerides, and LDL-C compared with healthy controls 46 .Discerning causality in observational research presents inherent difficulties, but the groundbreaking method of MR analysis has the potential to elucidate hypothetical causal relationships 17 .Unfortunately, a meticulous MR analysis here failed to reveal any substantial evidence for a causal relationship between the genetically determined levels of three circulating lipids and migraine.
Following the identification of a connection between circulating lipids and migraine in observational studies, researchers became intrigued by the potential therapeutic benefits of lipid-lowering medications in managing migraine.Statins are commonly used as prescription drugs in the field of neurology.Given the neurovascular nature of migraine, its etiology is influenced by inflammation and oxidative stress 47,48 , both regulated by statins 13,14 .The multifaceted properties of statins offer promising avenues for the advancement of migraine treatment.Furthermore, compelling evidence from animal studies suggests that statins may possess analgesic properties, further bolstering their potential as pain-relieving agents 49 .In a recent genetic association study conducted in a female migraine population, an intriguing link was discovered between migraines and specific lipoprotein subfractions, indicating a shared biological mechanism.The results of the colocalization analysis further identified this shared signal as circulating HMGCR, suggesting the potential effectiveness of statin analogs in the treatment of migraines 50 .Notably, exploring new applications of existing medications requires less time and resources than developing entirely new drugs de novo.In summary, statins have emerged as strong candidates for migraine treatment.Our MR study further supports this by highlighting the potential of HMGCR as a viable target for ameliorating migraine.Recent studies have focused primarily on the associations between statins, pain, and inflammation.For instance, in animal migraine models, atorvastatin suppressed B cell activation www.nature.com/scientificreports/ in the trigeminal caudal nucleus, demonstrating its ability to alleviate inflammation 49 .Similarly, in neuropathic pain models, pitavastatin demonstrated inhibitory effects on the JNK/P38/MAPK signaling pathway, resulting in a reduction in the release of inflammatory mediators 51 .
Nevertheless, existing research on the use of statins for migraine treatment presents conflicting results.While some case-control studies support the potential effectiveness of statins in alleviating migraine 52 , the results of several randomized controlled trials do not align with these results.These discrepancies could be attributed to several factors.First, the types of drugs used and their respective dosages were inconsistent across studies.Second, intervention measures within the control groups varied considerably.Third, the criteria for assessing migraine relief differed among studies.Finally, the durations of the interventions employed in the studies exhibited significant variation.Consequently, owing to the limited number of available studies, conducting a metaanalysis to quantitatively evaluate the efficacy of statins in the treatment of migraines would be premature.For further insight, please refer to Additional file 2, which provides a comprehensive overview of several published  www.nature.com/scientificreports/studies investigating the use of statins in migraine treatment.Based on current data, we find insufficient evidence to substantiate the use of statins as a definitive treatment for migraines.Further investigation is warranted to clarify the role of statins in migraine treatment across various layers of evidence, including genetic epidemiology.In this MR investigation, we used genetic variants associated with HMGCR expression and HMGCR-mediated circulating lipid levels as instrumental variables for statins.Primary and supplemental analyses consistently supported HMGCR inhibitors as having the potential to reduce the risk of migraine.Hence, our study has significant implications, warranting case-control and randomized controlled studies to evaluate whether statins prevent migraine.Additionally, it supports a causal connection between HMGCR and migraines.Further investigations should include diverse populations and establish animal models specifically targeting migraine to comprehensively explore the effects and mechanisms of action of statins on migraine management.
Our study had several strengths.First, we employed genetic variants as instrumental variables instead of directly utilizing statins as the exposure, effectively minimizing confounding factors and mitigating the influence of unmeasured biases.Additionally, we incorporated four distinct types of instrumental variable and crossvalidated our findings using data from two independent sources to enhance the reliability and robustness of our conclusions.Furthermore, to validate our instrumental variables, a positive control analysis was performed.
This study had some limitations.First, we were unable to identify suitable cis-acting pQTLs pertaining to HMGCR, which prevented us from establishing a clear association at the protein level between blood HMGCR levels and migraine.Second, despite the rigorous implementation of multiple sensitivity analyses to ensure that the MR assumptions were met, the presence of horizontal pleiotropy cannot be completely ruled out, which is an inherent limitation of MR studies.Third, the inclusion of populations of only European ancestry in the GWAS data restricts the generalizability of our findings.Therefore, caution should be exercised when extrapolating these associations to other populations.Fourth, we employed genetic variants in drug targets as proxies for the drug of interest rather than directly measuring drug-target inhibition, making direct comparisons challenging.Fifth, our analysis was limited to summary-level GWAS data, which restricted the depth of analysis of specific migraine subtypes.Finally, although the liver plays a crucial role in lipid metabolism, eQTLs data for this organ were not available.

Conclusion
The findings of this careful MR study imply a causative link between HMGCR inhibition and migraine.These results warrant clinical studies to assess the efficacy of HMGCR inhibition as a therapeutic strategy.Further investigations should elucidate the underlying protective mechanisms associated with this inhibition.

Figure 2 .
Figure 2. Associations linking HMG-CoA reductase gene expression or HMG-CoA reductase mediated lowdensity lipoprotein cholesterol, apolipoprotein B, and total cholesterol with the risk of migraine.

Figure 3 .
Figure 3.The visualization of HMG-CoA reductase gene expression and the colocalization analysis with migraine.

Figure 4 .
Figure 4. Meta-analysis of mendelian randomization analysis results based on inverse variance weighting.

Figure 5 .
Figure 5. Associations between genetically proxied statins lipid-lowering drugs and the risk of coronary artery disease as a positive control analysis.